PATHOGENICITY AND DIVERSITY OF VEGETATIVE COMPATIBILITY OF FUSARIUM VERTICILLIOIDES

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1 Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad, 113, , 2007 UDC 632.4: : Vesna S. Krnjaja 1, Jelena T. Leviã 2, Slavica Ÿ. Stankoviã 2, Zo rica M. To m iã 1 1 Institute for Animal Husbandry, Autoput 16, Belgrade, Republic of Serbia 2 Maize Research Institute, Zemun Polje, Slobodana Bajiãa 1, Belgrade, Republic of Serbia PATHOGENICITY AND DIVERSITY OF VEGETATIVE COMPATIBILITY OF FUSARIUM VERTICILLIOIDES ABSTRACT: Pathogenicity of 10 Fusarium verticillioides isolates, originated from grain of wheat (five isolates) and maize (five isolates), were studied under greenhouse conditions. Based on different parameters of the pathogenicity estimate (a scale for % of nonemerged plants, % of survived plants, plant vigour the growth and dry weight of roots and epicotyls and disease severity) it was determined that all F. verticillioides isolates expressed a different degree of pathogenicity. According to % of nonemerged plants six, three and one F. verticillioides isolates expressed low, moderate and high deegre of pathogenicity, respectively. All F. verticillioides isolates reduced the plant survival rate and vigour, while the disease severity ranged from 2.0 to Two types of nit mutants, nit1 and NitM, were obtained by the use of the method of vegetative compatibility. The frequency of nit1 mutants was greater (58.79%) than the frequency of NitM mutants (5.77%). A total of 10 vegetative compatibility groups (VCGs) of F. verticillioides were established in the complementation tests. These results point out to a high genetic diversity of F. verticillioides population. KEY WORDS: Fusarium verticillioides, pathogenicity, vegetative compatibility INTRODUCTION Fusarium verticillioides (Sacc.) Nirenberg (syn. Fusarium moniliforme Scheldon) is a widely distributed pathogen of maize (Zea mays L.) and many other plant species. In Serbia, F. verticillioides, as a pathogen of grain, was identified on maize, wheat and sorghum up to 77.8% (L e v i ã et al., 1997, 2003), 10% (D opuða and Leviã, 2004) and 7.5% (L e v i ã et al., 2006), respectively. The epidemiological studies show that F. verticillioides has one comparative advantage over other species of the genus Fusarium, especially in relation to F. graminearum Schwabe, as it requires a greater range of temperatures ( R e i d et al., 1999) and humidity (S c h n e i d e r and P e n d e r y 1983) for 103

2 its development, hence the competitiveness of the fungus will not change in different environments. It is typical of this species to colonise the plant tissue and to remain at the dormant stage or at the endophytic stage, as long as the tissue is healthy and active (M u n k v o l d and D e s j a r d i n s, 1997). It is difficult to discuss with certainty the role of F. verticillioides in the etiology of seedling diseases, as seedling infections and the disease development are produced by the seed affected by a disease or a contaminated soil, and depend on the temperature during the maize growing period (K o m m e - d a h l and W i n d e l s, 1981). F. verticillioides does not affect seed germination at the endophytic stage, but it affects the thickness, height, weight and leaf length of seedlings developed from infected seeds (Y a t e s et al., 1997). On the other hand, some strains of this fungus can even stimulate an earlier growth of seedlings. L e v i ã (2000) established that the frequency of occurrence of Fusarium species is not always correlated with their effects on seed germination. According to this author, the following species are most often isolated from maize grain: F. verticillioides (50.2%), F. subglutinans (Wollenw. & Reinking) Nelson (45.6%) and then F. proliferatum (Matsushima) Nirenberg (7.9%). However, germination of seeds infected with F. subglutinans, F. proliferatum and F. verticillioides amounted to 15.3%, 23.4% and 32.6%, respectively. The characterization of F. verticillioides isolates can be done on the basis of the seedling pathogenicity test or vegetative compatibility, since it was determined that isolates of the similar pathogenicity belonged to the same vegetative compatibility group (VCG) (K l e i n and C o r r e l l, 2001). Therefore, if a rapid method of the VCG identification is developed and rapid analyses of the population strain evaluation are provided, then the VCG pathogen strain identification can replace the pathogenicity test, which is time consuming and requires specific, control led conditions, depending on a plant species. Considering the economic importance of F. verticillioides, pathogenicity of F. verticillioides isolates, originating in maize and wheat grown at different locations in Serbia, to maize seedlings and their vegetative compatibility were observed in this study. MATERIAL AND METHODS Fungal isolates Ten isolates of F. verticillioides were used to perform pathogenicity and vegetative compatibility tests. Five isolates originated from grain of commercial maize hybrids grown in the vicinity of Belgrade Zemun, and five isolates originated from grain of wheat varieties grown at different locations in Serbia (Table 1). Isolates were identified as F. verticillioides using of the procedure outlined by N e l s o n et al. (1983) and B u r g e s s et al. (1994). 104

3 Tab. 1 F. verticilioides isolates tested for pathogenicity to maize seedlings under greenhouse conditions and vegetative compatibility No. Isolate Origin Host 1. MGA-7 Belgrade Zemun Commercial maize hybrid 2. MGD-4 Belgrade Zemun Commercial maize hybrid 3. MGE-5 Belgrade Zemun Commercial maize hybrid 4. MGG-13 Belgrade Zemun Commercial maize hybrid 5. MGI-1 Belgrade Zemun Commercial maize hybrid 6. MRIZP-201 Inðija Evropa 90 (wheat variety) 7. MRIZP-237 Inðija Pobeda (wheat variety) 8. MRIZP-570 Ruma Renesansa (wheat variety) 9. MRIZP-748 Loznica Simonida (wheat variety) 10. MRIZP-830 Sombor Evropa 90 (wheat variety) Selected cultures were initiated from single conidia and stored on PDA slants at 4 C, until use for the pathogenicity test and vegetative compatibility. Pathogenicity test with maize seedlings An insignificantly modified method described by M o l o t and S i m o - n e (1967) was followed for estimations of pathogenicity of F. verticillioides isolates. Petri dishes with the two-layer filter paper, instead of flasks, and sterile quartz sand, instead of soil, were used for the development of the fungus and artificial inoculation of seeds. A total of 45 maize seeds, surface-sterilised with sodium hypochlorite per isolate were inoculated in the sterile Petri dishes (ø 100 mm) with 30 ml of spore suspension (2 3 x 10 6 spore ml 1 ). The spore suspension was prepared from 7 10 old isolates cultured on the PDA at room temperature. Inoculated and non-inoculated (control) maize seeds were incubated at 22 C for two days and at 10 C for three days, and then planted into flats (40 x 18 x 16 cm) with sterile quartz sand, watered and incubated at C. Maize seeds were inoculated for two weeks and the following was determined: degree of pathogenicity, length (cm) and dry weight (g) of seedling roots and epicotyls. In this study, the degree of pathogenicity was defined on the basis of nonemerged plants (%), which was an outcome of seeds that had never germinated, and germinated seeds with completely rotted shoots. According to this parameter, the isolates were classified into five categories based on the scale described by M a ç k a (1989) (Table 2). Tab. 2 The scale for the estimation of pathogenicity of F. verticillioides isolates Percentage of nonemerged plants Degree of pathogenicity 0 10% not pathogenic 11 20% very low pathogenic 21 40% low pathogenic 41 60% moderate pathogenic 61 80% high pathogenic % very high pathogenic 105

4 Disease severity was also used as a measurement of pathogenicity of isolates, and was rated by a six-class scale, in which 0 = healthy root and epicotyl, and 5 = nongerminated seed, or completely rotted root and shoot. The length of each seedling from the seed attachment site to the top of the longest root and leaves was measured (cm). The detached root and epicotyl per replicate were dried at 60 C for 24 hours and then, their weights (g) were measured. Means were compared by Duncan's multiple range test. Vegetative compatibility groups Methods described by C o r r e l l et al. (1987) and K e d e r a et al. (1994) were used to isolate and characterize nit-mutants and their mutual pairing in order to determine vegetative compatibility of the studied F. verticillioides isolates. The excised pieces of mycelia were planted on the minimum medium (a basal medium amended with 30 g KClO 3, 2 g NaNO 3, and 1.6 g L-asparagine) for the selection of mutants (sectors). The basal medium contains 1.0 g KH 2 PO 4 ; 0.5 g MgSO 4 x7h 2 O; 0.5 g KCl; 10 mg FeSO 4 x7h 2 O; 0.2 ml sterile solution of microelemenats; 30.0 g of sucrose; 20.0 g of Difco agar; 1000 ml of distilled water. Pieces of hyphae and loose growing sectors were transferred to the basal medium with different nitrogen sources (NaNO 3, NaNO 2 and hypoxantine) in order to determine the type of the nit-mutant on the basis of a phenotype (P u h a l l a, 1985). Complementary nit1 and NitM mutants from each of 10 F. vertillicioides isolates were paired on the minimum medium (MM) in all possible combinations to perform complementation tests among the isolates. The nit mutants grew very sparsely across the medium, but complementation of auxotrophic mutants was indicated by a line of a vigorous growth where the mutants interacted. RESULTS AND DISCUSSION Pathogenicity All observed F. verticillioides isolates affected the survival rate and vigour of plants. Out of 10 F. verticillioides isolates tested under greenhouse conditions six, three and one isolates were low ( % of nonemerged plants), moderate ( % of nonemerged plants) and high (62.20% of nonemerged plants) pathogenic (Table 3). The isolate MGG-13, originated from maize grain, was estimated as high pathogenic as it reduced germination by 62.20%. The same isolate was significantly more pathogenic than the remaining isolates, as determined disease severity (3.69) was the highest and the survival rate (37.80%) and plant vigour were the lowest (Table 4). 106

5 Tab. 3 F. verticillioides isolates classified on the basis of a percentage of nonemerged plants Isolate Nonemerged plants (%) Degree of pathogenicity MGA low pathogenic MGD low pathogenic MGE low pathogenic MGG high pathogenic MGI moderate pathogenic MRIZP moderate pathogenic MRIZP low pathogenic MRIZP moderate pathogenic MRIZP low pathogenic MRIZP low pathogenic Control 4.47 The survival rate of seedlings developed from inoculated seeds with different isolates of F. verticillioides varied from 37.80% (MGG-13) to 73.33% (MGE-5), which was significantly lower than in the control (95.53%) (Table 4). The observed isolates affected the reduction of the root growth in comparison with the epicotyl growth. The isolate MGI-1, originated from maize, as well as, isolates MRIZP-201 and MRIZP-570, originated from wheat, expressed similar pathogenicity, which was particularly established on the basis of the root growth. The isolate MGD-4 showed peculiar behaviour, as disease severity caused to seedlings was high (3.18), which made it similar to the high pathogenic isolate (MGG-13), but due to a relatively high survival rate of plants (60.00%), it was estimated as low pathogenic (Table 4). Tab. 4 Effect of F. verticillioides isolates on maize seedlings growing from artificially infected seeds under greenhouse conditions No. Isolate Plant survival* (%) Plant vigour* Length (cm) Dry weight (g) Root Epicotyl Root Epicotyl Disease severity* 1. MGA bc bcd bc 2.10 bcd abc 2.66 bcd 2. MGD bcd cd bc 1.37 cde bc 3.18 abc 3. MGE ab b ab 2.23 bc abc 2.00 d 4. MGG d d 8.98 c 1.10 e bc 3.69 a 5. MGI cd d 9.87 c 1.33 de c 3.41 abc 6. MRIZP bcd d bc 1.00 e ab 3.54 ab 7. MRIZP bcd bc ab 2.00 bcd ab 2.54 bcd 8. MRIZP bcd d bc 1.23 de 0.56b c 3.02 abc 9. MRIZP bcd bcd bc 2.63 b bc 2.40 cd 10. MRIZP bcd d bc 1.80 bcde abc 2.48 cd Average Control a a a 3.97 a a 0.10 e LSD (0.05) LSD (0.01) * Values of column followed by the same letter(s) are not significantly different (P = 0.05) according to Duncan's multiple range test. 107

6 Our results on pathogenicity of F. verticillioides are in accordance with the results obtained by D e s j a r d i n s et al. (1995) and M unkvold and C a r l t o n (1997). Vegetative compatibility Mutants nit1 and NitM, with prevalence of nit1 (58.79%) over NitM (5.77%) (Table 5), were isolated from the observed isolates of F. verticillioides. According to the literature data (K littich and Leslie, 1988), the frequency of mutants nit1 is higher than the frequency of other types of nit mutants. Tab. 5 Frequency of nit1 and NitM mutants in the studied F. verticillioides isolates Isolate nit1 (%) NitM (%) MGA MGD MGE MGG MGI MRIZP MRIZP MRIZP MRIZP MRIZP Average Ten vegetative compatible groups (VCGs) of F. verticillioides were established on the basis of the complementation test among the isolates in all possible combinations. These results point out to a high genetic diversity of the population of this fungus pathogenic to maize. Similar results were stated by C h u l z e et al. (2000). CONCLUSION All studied F. verticillioides isolates originated from wheat (five isolates) and maize grain (five isolates) expressed pathogenicity to maize seedlings. According to the percentage of nonemerged plants, it was established that six, three and one isolates expressed low, moderate and high pathogenicity. The survival rate (%) and vigour (growth and dry weight of roots and epicotyls) of plants that were developed from inoculated seeds, were significantly reduced (approximately two times) in comparison with the control. There was a tendency for isolates from different hosts to have similar values for pathogenicity. These results are of a practical importance from the aspects of maize and wheat crop rotation and for the success of breeding for resistance to F. verticillioides. 108

7 The analysis of the results on pathogenicity obtained on the basis of the scale for the % nonemerged plants, plant survival rate (%), vigour (growth and dry weight of roots and epicotyls) and disease severity, shows a concurrence in defining moderate and high pathogenicity of isolates, while there was a certain nonconformance among these results in relation to low pathogenicity defining. Nevertheless, a selection of parameters, such as the scale for % of nonemerged plants, is a simple and good choice for the characterisation of pathogenicity degree of all F. verticillioides isolates. Two types of nit mutants, nit1 and NitM, were obtained by the use of the method of vegetative compatibility. The frequency of nit1 mutants was greater (58.79%) than the frequency of NitM mutants (5.77%). A total of 10 VCGs of F. verticillioides were determined in the complementation tests. This number of vegetative compatible groups indicates a high genetic diversity of the observed F. verticillioides population. ACKNOWLEDGEMENTS This paper is a part of the investigations implemented within the scope of the project No. TR-6826B financially supported by the Ministry of Science and Environmental Protection of the Republic of Serbia. REFERENCES Burgess, L. W., Summerell, B. A., Bullock, S., Gott, K. P., Backhouse, D. (1994): Laboratory for Fusarium Research, Third Edition, Fusarium Research Laboratory, Department of Crop Sciences, University of Sydney and Royal Botanic Garden, Sydney, 133. C h u l z e, S. N., R a m i r e z, M. L., T o r r e s, A., L e s l i e, J. F. (2000): Genetic variation in Fusarium Section Liseola from no-till maize in Argentina, Appl. Environ. Microbiol. 66 (12): Correll, J. C., Klittich, C. J. R., Leslie, J. F.(1987): Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests, Phytopathology 77: D e s j a r d i n s, A. E., P l a t t n e r, R. D., N e l s e n, T. C., L e s l i e, J. F. (1995): Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mais) seedlings, Appl. Environ. Microbiol. 61 (1): Dopuða, M., J. Leviã (2004): Sastav mikobiote (Fusaria) semena pšenice na podruåju Srema, Zb. radova str. 112, 5. Kong. z. bilja, Zlatibor, novembar Kedera, C. J., Leslie, J. F., Claflin, L. E. (1994): Genetic diversity of Fusarium section Liseola (Gibberella fujikuroi) in individual corn stalks, Phytopathology 84: K l e i n, K. K., C o r r e l l, J. C. (2001): Vegetative compatibility group diversity in Fusarium, Chapter 6, 83 96, in: Summerell, B. A., Leslie, J. F., Backhouse, D., 109

8 Bryden, W. L., Burgess, L. W. (ed.), Fusarium Paul E. Nelson Memorial Symposium, AS Press, The American Phytopathological Society, St. Paul, Minnesota, 392. Klittich, C. J. R., Leslie, J. F. (1988): Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi), Genetics 118: K o m m e d a h l, T., W i n d e l s, C. E. (1981): Root-, stalk-, and ear-infecting Fusarium species on corn in the USA, pp , in: Nelson, P. E., Toussoun, T. A., Cook, R. J. (eds), Fusarium: Diseases, Biology, and Taxonomy. The Pennsylvania State University Press, University Park and London, 457. L e v i ã, J. (2000): Uticaj Fusarium moniliforme, F. subglutinans i F. proliferatum na klijavost kukuruza, Zb. izvoda 120, Treãi jugoslovenski nauåno-struåni simpozijum iz selekcije i semenarstva (JUSEM). Zlatibor, 28. maja 1. juna Leviã, J., Ivanoviã, D., Stankoviã, S. (2003): Paraziti kukuruza, sirka i prosa koji se prenose semenom, B. lekar 6: Leviã, J., Tamburiã-Ilinåiã, Lj., Petroviã, T. (1997): Maize kernel infection by Fusarium species in the period , Cereal Res. Commun. 25 (3): M a ç k a, M. (1989): Fusaria as pathogens of cereal seedling, pp , in: Chelkowski, J. (ed.), Fusarium Mycotoxins, Taxonomy and Pathogenicity, Elsevier, Amsterdam Oxford New York Tokyo, 492. M o l o t, P. M., S i m o n e, J. (1967): Tehnique de contamination artificielle des semences de Mais pa les Fusarioses, Revie de Zoologie Agricole et Appliquee 1 3: Munkvold, G. P., Carlton, W. M. (1997): Influence of inoculation method on systemic Fusarium moniliforme infection of maize plants grown from infected seeds, Plant Dis. 81: Munkvold, G. P., Desjardins, A. E. (1997): Fumonisins in maize: can we reduce their occurrence? Plant Dis. 81: N e l s o n, P. E., T o u s s o u n, T. A., M a r a s a s, W. F. O. (1983): Fusarium species. An Illustrated Manual for Identification, The Pennsylvania State University Press, University Park and London, 193. P u h a l l a, J. E. (1985): Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility, Can. J. Bot. 63: Reid, L. M., Nicol, R. W., Ouellet, T., Stavard, M., Miller, J. D., Young, J. C., Stewart, D. W., Schaafsma, A. W. (1999): Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass and mycotoxin accumulation, Phytopathology 89: S c h n e i d e r, R. W., P e n d e r y, W. E. (1983): Stalk rot of corn: mechanism of predisposion by an early season water stress, Phytopathology 73: Y a t e s, I. E., B a c o n, C. W., H i n t o n, D. M. (1997): Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology, Plant Dis. 81:

9 PATOGENOST I DIVERZITET VEGETATIVNE KOMPATIBILNOSTI FUSARIUM VERTICILLIOIDES Vesna S. Krwaja 1, Jelena T. Leviã 2, Slavica Ÿ. Stankoviã 2, Zorica M. Tomiã 1 1 Institut za stoåarstvo, Auto put 16, 11081, Beograd Zemun, Srbija 2 Institut za kukuruz Zemun Poqe", Slobodana Bajiãa 1, 11185, Beograd, Srbija Rezime Patogenost 10 izolata F. verticillioides, poreklom iz zrna pšenice (5 izolata) i kukuruza (5 izolata), prouåavana je u uslovima staklenika. Na osnovu razliåitih parametara ocene patogenosti (skala za % neizniklih biqaka, % preÿivelih biqaka, vigora biqaka porast i suva teÿina korena i epikotila, i intenziteta bolesti) ustanovqeno je da su svi ispitivani izolati F. verticillioides ispoqili razliåit stepen patogenosti. Prema % neizniklih biqaka 6 izolata ispoqilo je nisku, 3 izolata sredwu i jedan izolat F. verticillioides visoku patogenost. Svi ispitivani izolati F. verticillioides su prouzrokovali smaweno preÿivqavawe i vigor biqaka, sa intenzitetom bolesti od 2.0 do Primenom metode vegetativne kompatibilnosti izolovane su dve vrste nit mutanata, nit1 i NitM. Uåestalost mutanata nit1 je bila veãa (58.79%) u odnosu na NitM mutante (5.77%). U komplementarnim testovima ustanovqeno je 10 vegetativno kompatibilnih grupa (VCG) F. verticillioides što ukazuje na visok genetiåki diverzitet populacije ovog patogena. 111